USEPA 2006 Perifiton Protocolos

8/11/2019 USEPA 2006 Perifiton Protocolos

1/23

DRAFT REVISIONSeptember 25, 1998

Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic

Macroinvertebrates, and Fish, Second Edition 6-1

6

PERIPHYTON PROTOCOLS

By R. Jan Stevenson, University of Louisville, and

Loren L. Bahls, University of Montana

Benthic algae (periphyton or phytobenthos) are primary producers and an important foundation ofmany stream food webs. These organisms also stabilize substrata and serve as habitat for many otherorganisms. Because benthic algal assemblages are attached to substrate, their characteristics areaffected by physical, chemical, and biological disturbances that occur in the stream reach during thetime in which the assemblage developed.

Diatoms in particular are useful ecological indicators because they are found in abundance in mostlotic ecosystems. Diatoms and many other algae can be identified to species by experiencedalgologists. The great numbers of species provide multiple, sensitive indicators of environmentalchange and the specific conditions of their habitat. Diatom species are differentially adapted to a widerange of ecological conditions.

Periphyton indices of biotic integrity have been developed and tested in several regions (KentuckyDepartment of Environmental Protection 1993, Hill 1997). Since the ecological tolerances for manyspecies are known (see section 6.1.4), changes in community composition can be used to diagnose theenvironmental stressors affecting ecological health, as well as to assess biotic integrity (Stevenson1998, Stevenson and Pan 1999).

Periphyton protocols may be used by themselves, but they are most effective when used with one ormore of the other assemblages and protocols. They should be used with habitat and benthicmacroinvertebrate assessments particularly because of the close relation between periphyton and theseelements of stream ecosystems.

Presently, few states have developed protocols for periphyton assessment. Montana, Kentucky, andOklahoma have developed periphyton bioassessment programs. Others states are exploring thepossibility of developing periphyton programs. Algae have been widely used to monitor water qualityin rivers of Europe, where many different approaches have been used for sampling and data analysis(see reviews in Whitton and Rott 1996, Whitton et al. 1991). The protocols presented here are acomposite of the techniques used in Kentucky, Montana, and Oklahoma (Bahls 1993, KentuckyDepartment of Environmental Protection 1993, Oklahoma Conservation Commission 1993).

Two Rapid Bioassessment Protocols for periphyton are presented. These protocols are meant toprovide examples of methods that can be used. Other methods are available and should be consideredbased on the objectives of the assessment program, resources available for study, numbers of streams

sampled, hypothesized stressors, and the physical habitat of the streams studied. Examples of othermethods are presented in textboxes throughout the chapter.

The first protocol (6.1) is a standard approach in which species composition and/or biomass of asampled assemblage is assessed in the laboratory. The second protocol (6.2) is a field-based rapidsurvey of periphyton biomass and coarse-level taxonomic composition (e.g., diatoms, filamentousgreens, blue-green algae) and requires little taxonomic expertise. The two protocols can be usedtogether. The first protocol has the advantage of providing much more accuracy in assessing biotic


2/23


3/23


4/23


6-4 Chapter 6: Periphyton Protocols

CHLOROPHYLL aSUBSAMPLING (OPTIONAL)

1. Chlorophyll asubsamples should be taken as soon aspossible (< 12 hours after sampling). Generally, ifchlorophyll subsamples can not be taken in the lab onthe day of collection, subsample in the field.

2. Homogenize samples. In the field, shake vigorously. In

the lab, use a tissue homogenizer.

3. Record the initial volume of sample on the periphytonsample log form.

4. Stir the sample on a magnetic stirrer and subsample.When subsampling, take at least two aliquots from thesample for each chlorophyll sample (two aliquots

provides a more representative subsample than one).Record the subsample volume for chlorophyll aon the

periphyton sample log form.

5. Concentrate the chlorophyll subsample on a glass fiberfilter (e.g., Whatman GFC or equivalent).

6. Fold the filter and wrap with aluminum to exclude light.

7. Store the filter in a cold cooler (not in water) andeventually in a freezer.

5. Place a permanent label on the outside of the sample container with the following information:waterbody name, location, station number, date, name of collector, and type of preservative.Record this information and relevant ecological information in a field notebook or on theperiphyton field data sheet (Appendix A-2, Form 1). Place another label with the same informationinside the sample container. (Caution! Lugol's solution and other iodine-based preservatives willturn paper labels black.)

6. After sampling, review the recorded information on all labels and forms for accuracy andcompleteness.

7. Examine all brushing and scraping tools for residues. Rub them clean and rinse them in distilledwater before sampling the next site and before putting them away.

8. Transport samples back to the laboratory in a cooler with ice (keep them cold and dark) and storepreserved samples in the dark until they are processed. Be sure to stow samples in a way so thattransport and shifting does not allow samples to leak. When preserved, check preservative everyfew weeks and replenish as necessary until taxonomic evaluation is completed.

9. Log in all incoming samples (Appendix A-2, Form 2). At a minimum, record sample identificationcode, date, stream name, sampling location, collector's name, sampling method, and area sampled(if it was determined).

6.1.1.2 Single Habitat Sampling

Variability due to differences in habitatbetween streams may be reduced bycollecting periphyton from a singlesubstrate/habitat combination thatcharacterizes the study reach (Rosen1995). For comparability of results,the same substrate/habitat combinationshould be sampled in all reference andtest streams. Single habitat samplingshould be used when biomass ofperiphyton will be assessed.

1. Define the sampling reach. Thearea sampled for single habitatsampling can be smaller than thearea used for multihabitat

sampling. Valuable results havebeen achieved in past projects bysampling just one riffle or pool.

2. Before sampling, complete thephysical/chemical field sheet (seeChapter 5; Appendix A-1, Form 1)and the periphyton field data sheet


5/23




QUALITY CONTROL (QC)

IN THE FIELD

1. Sample labels must be accurately andthoroughly completed, including the sampleidentification code, date, stream name,sampling location, and collector's name.The outside and any inside labels of thecontainer should contain the sameinformation. Chain of custody and samplelog forms must include the sameinformation as the sample container labels.

Caution! Lugol's solution and iodine-basedpreservatives will turn paper labels black.

2. After sampling has been completed at agiven site, all brushes, suction and scrapingdevices that have come in contact with thesample should be rubbed clean and rinsedthoroughly in distilled water. Theequipment should be examined again priorto use at the next sampling site, and rinsedagain if necessary.

3. After sampling, review the recordedinformation on all labels and forms foraccuracy and completeness.

4. Collect and analyze one replicate samplefrom 10% of the sites to evaluate precisionor repeatability of sampling technique,collection team, sample analysis, andtaxonomy.

(Appendix A-2, Form 1). Complete habitat assessments as in multihabitat sampling so that therelative importance of the habitats sampled can be characterized.

3. The recommended substrate/habitat combination is cobble obtained from riffles and runs withcurrent velocities of 10-50 cm/sec. Samples from this habitat are often easier to analyze than fromslow current habitats because they contain less silt. These habitats are common in many streams.

In low gradient streams where riffles are rare, algae on snags or in depositional habitats can becollected. Shifting sand is not recommended as a targeted substrate because the speciescomposition on sand is limited due to the small size and unstable nature of the substratum.Phytoplankton should be considered as an alternative to periphyton in large, low gradient streams.

4. Collect several subsamples from the same substrate/habitat combination and composite them into asingle container. Three or more subsamples should be collected from each reach or study stream.

5. The area sampled should always be determined if biomass (e.g., chlorophyll) per unit area is to bemeasured.

6. If you plan to assay samples for chlorophyll a,

do not preserve samples until they have beensubsampled (see textbox entitled ChlorophyllaSubsampling).

7. Store, transport, process, and log in samples asin steps 4-9 in section 6.1.1.1.

6.1.2 Field Sampling Procedures:

Artificial Substrates

Most monitoring groups prefer sampling natural

substrates whenever possible to reduce field timeand improve ecological applicability of information.However, periphyton can also be sampled bycollecting from artificial substrates that are placedin aquatic habitats and colonized over a period oftime. This procedure is particularly useful innon-wadeable streams, rivers with no riffle areas,wetlands, or the littoral zones of lentic habitats.Both natural and artificial substrates are useful inmonitoring and assessing waterbody conditions, andhave corresponding advantages and disadvantages(Stevenson and Lowe 1986, Aloi 1990). The

methods summarized here are a composite of thosespecified by Kentucky (Kentucky DEP 1993),Florida (Florida DEP 1996), and Oklahoma(Oklahoma CC 1993).Although glass microslides are preferred, a varietyof artificial substrates have been used with success(see #2 below and textbox on p 6-6).


6/23



FIELD EQUIPMENT/SUPPLIES NEEDED FOR

PERIPHYTON SAMPLING--

ARTIFICIAL SUBSTRATES

C periphytometer (frame to hold artificial substrata)C microslides or other suitable substratum (e.g.,

clay tiles, sanded Plexiglass plates, or woodenor acrylic dowels)

C sledge hammer and rebarsC toothbrush, razor blade, or other scraping toolsC water bottle with distilled waterC white plastic or enamel panC aluminium foilC sample containersC sample container labels

C field notebook (waterproof)C preservative [Lugol's solution, 4% bufferedformalin, "M3" fixative, or 2% glutaraldehyde(APHA 1995)]

C cooler with ice

1. Microslides should be thoroughly cleaned before placing in periphytometers (e.g., Patrick et al.1954). Rinse slides in acetone and clean with Kimwipes.

2. Place surface (floating) or benthic (bottom) periphytometers fitted with glass slides, glass rods,clay tiles, plexiglass plates or similar substrates in the study area. Allow 2 to 4 weeks forperiphyton recruitment and colonization.

3. Replicate a minimum of 3 periphytometers at each site to account for spatial variability. The totalnumber should depend upon the study design and hypotheses tested. Samples can either becomposited or analyzed individually.

4. Attach periphytometers to rebars pounded into the stream bottom or to other stable structures.Periphytometers should be hidden from view to minimize disturbance or vandalism. Avoid themain channel of floatable, recreational streams. Each periphytometer should be oriented with theshield directed upstream.

5. If flooding or a similar scouring eventoccurs during incubation, allow waterbodyto equilibrate and reset periphytometers

with clean slides.

6. After the incubation period (2-4 weeks),collect substrates. Remove algae usingrubber spatulas, toothbrushes and razorblades. You can tell when all algae havebeen removed from substrates by a changefrom smooth, mucilaginous feel (evenwhen no visible algae are present) to anon-slimy or rough texture.

7. Store, transport, process, and log insamples as in steps 4-9 in section 6.1.1.1.

8. One advantage of using artificialsubstrates is that containers (e.g.,whirl-pack bags or sample jars) can bepurchased that will hold the substrates sothat substrates need not be scraped in the field. Different substrates can be designated formicroscopic analysis and chlorophyll assay. Then algae and substrates can be placed in samplingcontainers and preserved for later processing and microscopic analysis or placed in a cooler on icefor later chlorophyll aanalysis. Laboratory sample processing is preferred; so if travel and holdingtime are less than 12 hours, it is not necessary to split samples before returning to the lab.

6.1.3 Assessing Relative Abundances of Algal Taxa: Both "Soft" (Non-Diatom)

Algae and Diatoms

The Methods summarized here are a modified version of those used by Kentucky (Kentucky DEP1993), Florida (Florida DEP 1996), and Montana (Bahls 1993). For more detail or for alternativemethods, see Standard Methods for the Examination of Water and Wastewater (APHA 1995).

Many algae are readily identifiable to species level by trained personnel who have a good library of


7/23




literature on algal taxonomy (see section 6.3). All algae can not be identified to species because: thegrowth forms of some algal species are morphologically indistinguishable with the light microscope(e.g., zoospores of many green algae); the species has not been described previously; or the species isnot in the laboratorys literature. Consistency in identifications within a laboratory and program isvery important, because most bioassessment are based on contrasts between reference and test sites.Accuracy of identifications becomes most important when using autecological information from other

studies. Quality assurance techniques are designed to ensure "internal consistency" and also improvecomparisons with information in other algal assessment and monitoring programs.

6.1.3.1 "Soft" (Non-Diatom) Algae Relative Abundance and Taxa Richness

1. Homogenize algal samples with a tissue homogenizer or blender.

2. Thoroughly mix the homogenized sample and pipette into a Palmer counting cell (see textbox foralternative methods). Algal suspensions that produce between 10 and 20 cells in a field providegood densities for counting and identifying cells. Lower densities slow counting. Dilute samples ifcells overlap too much for counting.

3. Fill in the top portion of the benchsheet for "soft" algae (Appendix A-2, Form 3) with enoughinformation from the sample label and other sources to uniquely identify the sample.

4. Identify and count 300 algal "cell units" to the lowest possible taxonomic level at 400Xmagnification with the use of the references in Section 6.3.

! Distinguishing cells of coenocytic algae (e.g., Vaucheria) and small filaments of blue-greenalgae is a problem in cell counts. "Cell units" can be defined for these algae as 10mm sectionsof the thallus or filament.

! For diatoms, only count live diatoms and do not identify to lower taxonomic levels if asubsequent count of cleaned diatoms is to be undertaken (See section 6.1.3.2).

! Record numbers of cells or cell units observed for each taxon on a benchsheet.! Make taxonomic notes and drawings on benchsheets of important specimens.

5. Optional - To better determine non-diatom taxa richness, continue counting until you have not

observed any new taxa for 100 cell units or about three minutes of observation.

6.1.3.2 Diatom Relative Abundances and Taxa Richness

1. Subsample at least 5-10 mL of concentrated preserved sample while vigorously shaking the sample(or using magnetic stirrer). Oxidize (clean) samples for diatom analysis (APHA 1995, see textboxentitled Oxidation Methods for Cleaning Diatoms).

2. Mount diatoms in Naphrax or another high refractive index medium to make permanent slides.Label slides with same information as on the sample container label.

3. Fill in the top portion of the bench sheet for diatom counts (Appendix A-2, Form 4) with enoughinformation from the sample label to uniquely identify the sample.

4. Identify and count diatom valves to the lowest possible taxonomic level, which should be species

and perhaps variety level, under oil immersion at 1000X magnification with the use of the


8/23



references in Section 6.3. At minimum, count 600 valves (300 cells) and at least until 10 valves of10 species have been observed. Be careful to distinguish and count both valves of intact frustules.The 10 valves of 10 species rule ensures relatively precise estimates of relative abundances of thedominant taxa when one or two taxa are highly dominant. Six hundred valve counts were chosento conform with methods used in other national bioassessment programs (Porter et al. 1993).Record numbers of valves observed for each taxon on the bench sheet. Make taxonomic notes and

drawings on benchsheets and record stage coordinates of important specimens.

5. Optional - To estimate total diatom taxa richness, continue counting until you have not observedany new species for 100 specimens or about three minutes of observation.

6.1.3.3 Calculating Species Relative Abundances and Taxa Richness

1. Relative abundances of "soft" algae are determined by dividing the number of cells (cell units)counted for each taxon by the total number of cells counted (e.g., 300). Enter this information onAppendix A-2, Form 3.

2. Relative abundances of diatoms have to be corrected for the number of live diatoms observed in the

count of all algae. Therefore, determine the relative abundances of diatom species in the algalassemblage by dividing the number of valves counted for each species by the total number ofvalves counted (e.g., 600); then multiply the relative abundance of each diatom taxon in the diatomcount by the relative abundance of live diatoms in the count of all algae. Enter this information onAppendix A-2, Form 4. Some analysts prefer to treat diatom and soft algal species compositionseparately. In this case, determine the relative abundances of diatom species in the algalassemblage by dividing the number of valves counted for each species by the total number ofvalves counted (e.g., 600).

3. Total taxa richness can be estimated by adding the number of "soft" algal taxa and diatom taxa.

6.1.3.4 Alternative Preparation Techniques

Palmer counting cells are excellent for identifying and counting soft-algae in most species assemblages.When samples have many very small blue-green algae or a few, relatively important large cells, otherslide preparation techniques may be useful to increase magnification and sample size, respectively.Because accurate diatom identification is not possible in Palmer cells, we have recommended countingcleaned diatoms in special mounts. However, if the taxonomy of algae in samples is well known,preparation and counting time can be reduced by mounting algae in syrup. In syrup, both soft algaeand diatoms can be identified, but resolution of morphological details of diatoms is not as great as inmounts of diatoms in resins (e.g., Naphrax).

Assemblages with many small cells:We recommend a simple wet mount procedure when samples

contain many small algae so samples can be observed at 1000X. A small volume of water under thecoverglass prevents movement of cells when adjusting focus and using oil immersion. Thesepreparations usually last several days if properly sealed (see below).

Wet mounts:1. Clean coverglasses and place on flat surface.

2. Pipette 1.0 mL of algal suspension onto the coverglass.


9/23


10/23



3. Place 1.0 mL of algal suspension on coverglass. Consider using several dilutions.

4. Let dry for 24 hours. Alternatively, dry on slide warmer on low setting. Do not overdry or cellswill plasmolyze.

5. Place another .1.0 mL of 10% TSM on cover glass and dry (overnight or 4 hours on a slidewarmer). Apply 10% TSM quickly to avoid patchy resuspension of the original layer of TSM and

algae.

6. Invert coverglass onto microscope slide; place slide on hot plate to warm the slide and syrup. Donot boil, just warm. Press coverglass gently in place with forceps, being careful to keep all syrupunder the coverglass. The syrup should spread under coverglass.

7. Remove the slide from the hotplate. Cooling should partially seal the coverglass to the slide.

8. More permanently seal the syrup under slides by painting fingernail polish around the edge of thecover glass and onto the microscope slide.

Note: Preserve color of chloroplasts by keeping samples in dark.

Special Note: If slides get too warm in storage, syrup will loose viscosity and become runny. Algaeand medium may then escape containment under coverglass. Store slides in a horizontal position.

6.1.4 Metrics Based on Species Composition

The periphyton metrics presented here are used by several states and environmental assessmentprograms throughout the US and Europe (e.g., Kentucky DEP 1993, Bahls 1993, Florida DEP 1996,Whitton et al. 1991, Whitton and Kelly 1995). Each of these metrics should be tested for response tohuman alterations of streams in the region in which they are used (see Chapter 9, Biological DataAnalysis). In many cases, diatom and soft algal metrics have been determined separately because

changes in small abundant cyanobacteria (blue-green algae) can numerically overwhelm metrics basedon relative abundance and because green algae with large cells (e.g., Cladophora) may not haveappropriate weight. However, attempts should be made to integrate diatoms and soft algae in as manymetrics as possible, especially in cases such as species and generic richness when great variability inrelative abundance is not an issue.


11/23




COSTS AND BENEFITS OF SIMPLER

ANALYSES

We recommend that all algae (soft and diatom)be identified and counted. Information may belost if soft algae are not identified and counted

because some impacts may selectively affect softalgae. Most of the species (and thusinformation) in a sample will be diatoms. Costsof both analyses are not that great.

Costs can be reduced by only counting diatomsor soft algae. Since diatoms are usually themost species-rich group of algae in samples andmost metrics are based on differences intaxonomic composition, we recommend thatdiatoms be counted. In addition, permanently

preserved and readily archived microslides ofdiatoms can serve as a historic reference of

ecological conditions.

In general, identifying algae to species isrecommended for two reasons: (1) to bettercharacterize differences between assemblagesthat may occur at the species level and (2)

because large differences in ecologicalpreferences do exist among algal species withinthe same genus.

However, substantial information can be gainedby identifying algae just to the genus level.Whereas identifying algae only to genus may

loose valuable ecological information, costs ofanalyses can be reduced, especially forinexperienced analysts.

If implementing a new program and only aninexperienced analyst is available for the job,identifying diatom genera in assemblages can

provide valuable characterizations of bioticintegrity and environmental conditions.

As analysts get more experience counting, thetaxonomic level of their analyses shouldimprove. The cost of an experienced analystcounting and identifying algae to species is notmuch greater than analysis to genus.

Many metrics can be calculated based onpresence/absence data or on relativeabundances of taxa. For example, percentPollution Tolerant Diatoms can be calculatedas the sum of relative abundances of pollutiontolerant taxa in an assemblage or as the

number of species that are tolerant to pollutionin an assemblage. Percent communitysimilarity can be calculated as presentedbelow, which quantifies the percent oforganisms in two assemblages that are thesame. Alternatively, it can be calculated as thepercent of species that are the same by makingall relative abundances greater than 0 equal to1. The following metrics can also becalculated with presence/absence data insteadof species relative abundances: % sensitivetaxa, % motile taxa, % acidobiontic, %

alkalibiontic, % halobiontic, % saprobiontic,% eutrophic, simple autecological indices, andchange in inferred ecological conditions.Although we may find that metrics based onspecies relative abundances are more sensitiveto environmental change, metrics based onpresence/absence data may be moreappropriate when developing metrics withmultihabitat samples and proportionalsampling of habitats is difficult. In the lattercase, presence/absence of species shouldremain the same, even if relative abundance oftaxa differs with biases in multihabitatsampling.

The metrics have been divided into two groupswhich may be helpful in developing an Indexof Biotic Integrity (IBI). Metrics in the firstgroup are less diagnostic than the secondgroup of metrics. Metrics in the first group(species and generic richness, Shannondiversity, etc.) generally characterize bioticintegrity ("natural balance in flora and

fauna." as in Karr and Dudley 1981)without specifically diagnosing ecologicalconditions and causes of impairment. Thesecond group of metrics more specificallydiagnoses causes of impaired biotic integrity.Metrics from both groups could be included in an IBI to make a hierarchically diagnostic IBI.Alternatively, an IBI could be constructed from only metrics of biotic integrity so that inference ofbiotic integrity and diagnosis of impairment are independent (Stevenson and Pan 1999).


12/23


1David Beeson is a phycologist with Schafer & Associates, Inc.


Autecological information about many algal species and genera has been reported in the literature.This information comes in several forms. In some cases, qualitative descriptions of the ecologicalconditions in which species were observed were reported in early studies of diatoms. Following thedevelopment of the saprobic index by Kolkwitz and Marsson (1908), several categorical classificationsystems (e.g., halobian spectrum, pH spectrum) were developed to describe the ecological preferencesand tolerances of species (see Lowe 1974 for a review). Most recently, the ecological optima and

tolerances of species for specific environmental conditions have been quantified by using weightedaverage regression approaches (see ter Braak and van Dam 1989 for a review). We have compiled alist of references for this information in Section 6.4. These references will be valuable for developingmany of the metrics below.

Metrics of Biotic Integrity

1. Species richnessis an estimate of the number of algal species (diatoms, soft algae, or both) ina sample. High species richness is assumed to indicate high biotic integrity because manyspecies are adapted to the conditions present in the habitat. Species richness is predicted todecrease with increasing pollution because many species are stressed. However, many habitatsmay be naturally stressed by low nutrients, low light, or other factors. Slight increases in

nutrient enrichment can increase species richness in headwater and naturally unproductive,nutrient-poor streams (Bahls et al. 1992).

2. Total Number of Genera(Generic richness) should be highest in reference sites and lowest inimpacted sites where sensitive genera become stressed. Total number of genera (diatoms, softalgae, or both) may provide a more robust measure of diversity than species richness, becausenumerous closely related species are within some genera and may artificially inflate richnessestimates.

3. Total Number of Divisionsrepresented by all taxa should be highest in sites with good waterquality and high biotic integrity.

4. Shannon Diversity (for diatoms). The Shannon Index is a function of both the number ofspecies in a sample and the distribution of individuals among those species (Klemm et al.1990). Because species richness and evenness may vary independently and complexly withwater pollution. Stevenson (1984) suggests that changes in species diversity, rather than thediversity value, may be useful indicators of changes in water quality. Species diversity,despite the controversy surrounding it, has historically been used with success as an indicatorof organic (sewage) pollution (Wilhm and Dorris 1968, Weber 1973, Cooper and Wilhm1975). Bahls et al. (1992) uses Shannon diversity because of its sensitivity to water qualitychanges. Under certain conditions Shannon diversity values may underestimate water qualitye.g., when total number of taxa is less than 10. Assessments for low richness samples can beimproved by comparing the assemblage Shannon Diversity to the Maximum Shannon Diversity

value (David Beeson1, personal communication).

5. Percent Community Similarity (PSc) of Diatoms. The percent community similarity (PSc)index, discussed by Whittaker (1952), was used by Whittaker and Fairbanks (1958) tocompare planktonic copepod communities. It was chosen for use in algal bioassessmentbecause it shows community similarities based on relative abundances, and in doing so, gives


13/23




PSc ' 100&.5Esi'1*ai&bi* ' E

si'1min(ai,bi)

PTI 'EnitiN

more weight to dominant taxa than rare ones. Percent similarity can be used to comparecontrol and test sites, or average community of a group of control or reference sites with a testsite. Percent community similarity values range from 0 (no similarity) to 100%.

The formula for calculating percent community similarity is:

where:

ai= percentage of species i in sample Abi= percentage of species i in sample B

6. Pollution Tolerance Index for Diatoms. The pollution tolerance index (PTI) for algaeresembles the Hilsenhoff biotic index for macroinvertebrates (Hilsenhoff 1987). Lange-Bertalot (1979) distinguishes three categories of diatoms according to their tolerance toincreased pollution, with species assigned a value of 1 for most tolerant taxa (e.g., Nitzschia

paleaor Gomphonema parvulum) to 3 for relatively sensitive species. Relative tolerance fortaxa can be found in Lange-Bertalot (1979) and in many of the references listed in section 6.4.Thus, Lange-Bertalots PTI varies from 1 for most polluted to 3 for least polluted waters whenusing the following equation:

where:

ni= number of cells counted for species iti = tolerance value of species iN = total number of cells counted

In some cases, the range of values for tolerances has been increased, thereby producing acorresponding increase in the range of PTI values.

7. Percent Sensitive Diatoms. The percent sensitive diatoms metric is the sum of the relativeabundances of all intolerant species. This metric is especially important in smaller-orderstreams where primary productivity may be naturally low, causing many other metrics tounderestimate water quality.

8. Percent Achnanthes minutissima. This species is a cosmopolitan diatom that has a very

broad ecological amplitude. It is an attached diatom and often the first species to pioneer arecently scoured site, sometimes to the exclusion of all other algae. A. minutissimais alsofrequently dominant in streams subjected to acid mine drainage (e.g., Silver Bow Creek,Montana) and to other chemical insults. The percent abundance ofA. minutissimahas beenfound to be directly proportional to the time that has elapsed since the last scouring flow orepisode of toxic pollution. For use in bioassessment, the quartiles of this metric from a


14/23



population of sites has been used to establish judgment criteria, e.g., 0-25% = no disturbance,25-50% = minor disturbance, 50-75% = moderate disturbance, and 75-100% = severedisturbance. Least-impaired streams in Montana may contain up to 50%A. minutissima(Bahls, unpublished data).

9. Percent live diatomswas proposed by Hill (1997) as a metric to indicate the health of the

diatom assemblage. Low percent live diatoms could be due to heavy sedimentation and/orrelatively old algal assemblages with high algal biomass on substrates.

Diagnostic Metrics that Infer Ecological Conditions

The ecological preferences of many diatoms and other algae have been recorded in the literature. Usingrelative abundances of algal species in the sample and their preferences for specific habitat conditions,metrics can be calculated to indicate the environment stressors in a habitat. These metrics can morespecifically infer environmental stressors than the general pollution tolerance index.

10. Percent Aberrant Diatomsis the percent of diatoms in a sample that have anomalies in striaepatterns or frustule shape (e.g, long cells that are bent or cells with indentations). This metric

has been positively correlated to heavy metal contamination in streams (McFarland et al.1997).

11. Percent Motile Diatoms. The percent motile diatoms is a siltation index, expressed as therelative abundance ofNavicula+Nitzschia+ Surirella. It has shown promise in Montana(Bahls et al. 1992). The three genera are able to crawl towards the surface if they are coveredby silt; their abundance is thought to reflect the amount and frequency of siltation. Relativeabundances of Gyrosigma, Cylindrotheca, and other motile diatoms may also be added to thismetric.

12. Simple Diagnostic Metricscan infer the environmental stressor based on the autecology of

individual species in the habitats. For example, if acid mine drainage was impairing streamconditions, then we would expect to find more acidobiontic taxa in samples. Calculate asimple diagnostic metric as the sum of the percent relative abundances (range 0-100%) ofspecies that have environmental optima in extreme environmental conditions. For example (seeTable 6-2):

% acidobiontic + % acidophilic

% alkalibiontic + % alkaliphilic

% halophilic

% mesosaprobic + % oligosaprobic + % saprophilic

% eutrophic

13. Inferred Ecological Conditions with Simple Autecological Indices (SAI)- The ecologicalpreferences for diatoms are commonly recorded in the literature. Using the standard ecologicalcategories compiled by Lowe (1974, Table 6-2), the ecological preferences for different diatomspecies can be characterized along an environmental (stressor) gradient. For example, pHpreferences for many taxa are known. These preferences (1i) can be ranked from 1-5 (e.g.,acidobiontic, acidophilic, indifferent, alkaliphilic, alkalibiontic, Table 6-2) and can be used inthe following equation to infer environmental conditions (EC) and effect on the periphytonassemblage.


15/23


16/23


Classification System/

Ecological Parameter Class Subclass

Conditions of Highest Relative

Abundances


Mesotrophic Moderate nutrient conditions

Oligotrophic Low nutrient conditions

Dystrophic High humic (DOC) conditions

Halobion Spectrum - basedon chloride concentrations orconductivity

Polyhalobous Salt concentrations > 40,000 mg/L

Euhalobous Marine forms: 30,000-40,000 mg/L

Mesohalobous Alpha range Brackish water forms: 10,000-30,000 mg/L

Mesohalobous Beta range Brackish water forms: 500-10,000 mg/L

Oligohalobous Halophilous Freshwater - stimulated by some salt

Oligohalobous Indifferent Freshwater - tolerates some salt

Oligohalobous Halophobic Freshwater - does not tolerate smallamounts of salt

Saprobien System - based onorganic pollution

Polysaprobic Characteristic of zone of degradation andputrefication, oxygen usually absent or low

in concentrationMesosaprobic Alpha range Zone of organic load oxidation N as

amino acids

Beta range Zone of organic load oxidation N asammonia

Oligosaprobic Zone in which oxidation of organicscomplete, but high nutrient concentrations

persist

Saprophilic Usually in polluted waters, but also inclean waters

Saproxenous Usually in clean waters, but also found inpolluted waters

Saprophobic Only found in unpolluted waters

6.1.5 Determining Periphyton Biomass

Measurement of periphyton biomass is common in many studies and may be especially important instudies that address nutrient enrichment or toxicity. In many cases, however, sampling benthic algaemisses peak biomass, which may best indicate nutrient problems and potential for nuisance algalgrowths (Biggs 1996, Stevenson 1996).

Biomass measurements can be made with samples collected from natural or artificial substrates. To

quantify algal biomass (chl a, ash-free dry mass, cell density, biovolume cm-2), the area of the substratesampled must be determined. Two national stream assessment programs sample and assessarea-specific cell density and biovolume (USGS-NAWQA, Porter et al. 1993; and EMAP, Klemm andLazorchak 1994). These programs estimate algal biomass in habitats and reaches by collectingcomposite samples separately from riffle and pool habitats.


17/23




LABORATORY EQUIPMENT FOR

PERIPHYTON ANALYSIS

compound microscope with 10X or 15Xoculars and 20X, 40X and 100X (oil)objectives

tally counter (for species proportional count) microscope slides and coverglasses immersion oil, lens paper and absorbent

tissues tissue homogenizer or blender magnetic stirrer and stir bar forceps hot plate fume hood squeeze bottle with distilled water oxidation reagents (HNO3, H2SO4, K2Cr2O7,

H2O2) 200-500 ml beakers safety glasses and protective clothing drying oven for AFDM muffle furnace for AFDM aluminum weighing pans for AFDM spectrophotometer or fluorometer for chla centrifuge for chl a graduated test tubes for chl a acetone for chl a MgCO3for chl a

Periphyton biomass can be estimated with chla, ash-free dry mass (AFDM), cell densities,and biovolume, usually per cm2(Stevenson1996). Each of these measures estimates adifferent component of periphyton biomass (seeStevenson 1996 for discussion).

6.1.5.1 Chlorophyll a

Chlorophyll a ranges from 0.5 to 2% of totalalgal biomass (APHA 1995), and this ratiovaries with taxonomy, light, and nutrients. Adetailed description of chlorophyll aanalysis isbeyond the scope of this chapter. Standardmethods (APHA 1995, USEPA 1992) arereadily available. The analysis is relativelysimple and involves:

1. extracting chlorophyll ain acetone;

2. measuring chlorophyll concentration in theextract with a spectrophotometer orfluorometer; and

3. calculating chlorophyll density onsubstrates by determining the proportion oforiginal sample that was assessed forchlorophyll.

6.1.5.2 Ash-Free Dry Mass

Ash-free dry mass is a measurement of the organic matter in samples, and includes biomass ofbacteria, fungi, small fauna, and detritus in samples. A detailed description of analysis is beyond thescope of this chapter, but standard methods (APHA 1995, USEPA 1995) are readily available. Theanalysis is relatively simple and measures the difference in mass of a sample after drying and afterincinerating organic matter in the sample. We recommend using AFDM versus dry mass to measureperiphyton biomass because silt can account for a substantial proportion of dry mass in some samples.Ash mass in samples can be used to infer the amount of silt or other inorganic matter in samples.

6.1.5.3 Area-Specific Cell Densities and Biovolumes

Cell densities (cells cm-2) are determined by dividing the numbers of cells counted by the proportion ofsample counted and the area from which samples were collected. Cell biovolumes (mm3biovolumecm-2) are determined by summing the products of cell density and biovolume of each species counted(see Lowe and Pan 1996) and dividing that sum by the proportion of sample counted and the area fromwhich samples were collected.


18/23



QUALITY CONTROL IN THE LABORATORY

1. Upon delivery of samples to the laboratory, completeentries on periphyton sample log-in forms (Appendix 2,Form 2).

2. Maintain a voucher collection of all samples and diatomslides. They should be accurately and completely labeled,preserved, and stored in the laboratory for futurereference. Specimens on diatom slides should be clearlycircled with a diamond or ink marker to facilitatelocation. A record of the voucher specimens should bemaintained. Photographs of specimens improve"in-house" QA.

3. For every QA/QC sample (replicate sample in every 10thstream), assess relative abundances and taxa richness inreplicate wet mounts and a replicate diatom slide to assessvariation in metrics due to variability in sampling within

reaches (habitats), sample preparation, and analyticalvariability.

4. QA/QC samples should be counted by another taxonomistto assess taxonomic precision and bias, if possible.

5. Common algal taxa should be the same for the two wetmount replicates. The percent community similarityindex (Whittaker 1952) (see Section 6.5.1) calculatedfrom proportional counts of the two replicate diatomslides should exceed 75%.

6. If it is not possible to get another taxonomist in the lab toQA/QC samples, an outside taxonomist should beconsulted on a periodic basis to spot-check and verifytaxonomic identifications in wet mounts and diatomslides. All common genera in the wet mount and allmajor species on the diatom slide (>3% relativeabundance) should be identified similarly by both analysts(synonyms are acceptable). Any differences inidentification should be reconciled and bench sheetsshould be corrected.

7. A library of basic taxonomic literature is an essential aidin the identification of algae and should be maintained

and updated as needed in the laboratory (see taxonomicreferences for periphyton in Section 6.5). Taxonomistsshould participate in periodic training to ensure accurateidentifications

6.1.5.4 Biomass Metrics

High algal biomass can indicateeutrophication, but high algalbiomass can also accumulate in lessproductive habitats after long

periods of stable flow. Low algalbiomass may be due to toxicconditions, but could be due to arecent storm event and spate ornaturally heavy grazing. Thus,interpretation of biomass results isambiguous and is the reason thatmajor emphasis has not been placedon quantifying algal biomass forRBP. However, nuisance levels ofalgal biomass (e.g., > 10 g chl acm-2, > 5 mg AFDM cm-2, > 40%

cover by macroalgae; see review byBiggs 1996) do indicate nutrient ororganic enrichment. If repeatedmeasurements of biomass can bemade, then the mean and maximumbenthic chl acould be used to definetrophic status of streams. Dodds etal. (1998) have proposed guidelinesin which theoligotrophic-mesotrophic boundaryis a mean benthic chl aof 2 g cm-2

or a maximum benthic chl aof 7 gcm-2and the mesotrophic-eutrophicboundary is a mean of 6 g chl acm-2and a maximum of 20 g chl acm-2.

6.2 FIELD-BASED RAPID

PERIPHYTON SURVEY

Semi-quantitative assessments ofbenthic algal biomass and taxonomiccomposition can be made rapidly

with a viewing bucket marked with agrid and a biomass scoring system.The advantage of using thistechnique is that it enables rapidassessment of algal biomass overlarger spatial scales than substrate sampling and laboratory analysis. Coarse-level taxonomiccharacterization of communities is also possible with this technique. This technique is a survey of the


19/23


20/23



5. Statistically characterize density of algae on substrate by determining:! total number of grid points (dots) evaluated at the site (Dt);! number of grid points (dots) over macroalgae (Dm)! total number of grid points (dots) over suitable substrate for microalgae at the site (dt);! number of grid points over microalga of different thickness ranks for each type of microalga

(di);! average percent cover of the habitat by each type of macroalgae (i.e., 100X Dm/Dt);! maximum length of each type of macroalgae;! mean density (i.e., thickness rank) of each type of macroalgae on suitable substrate (i.e.,

Ediri/dt); maximum density of each type of microalgae on suitable substrate.

6. QA/QC between observers and calibration between algal biomass (chl a, AFDM, cell density andbiovolume cm-2and taxonomic composition) can be developed by collecting samples that havespecific microalgal rankings and assaying the periphyton.

6.3 TAXONOMIC REFERENCES FOR PERIPHYTON

A great wealth of taxonomic literature is available for algae. Below is a subset of that literature. It isa list of taxonomic references that are useful for most of the United States and are either in English, areimportant because no English treatment of the group is adequate, or are valuable for the goodillustrations.

Camburn, K.E., R.L. Lowe, and D.L. Stoneburner. 1978. The haptobenthic diatom flora of LongBranch Creek, South Carolina.Nova Hedwigia30:149-279.

Collins, G.B. and R.G. Kalinsky. 1977. Studies on Ohio diatoms: I. Diatoms of the Scioto RiverBasin. Bull. Ohio Biological Survey. 5(3):1-45.

Cox, E. J. 1996. Identification of freshwater diatoms from live material. Chapman & Hall, London.

Czarnecki, D.B. and D.W. Blinn. 1978. Diatoms of the Colorado River in Grand Canyon NationalPark and vicinity. (Diatoms of Southwestern USA II). Bibliotheca Phycologia 38. J. Cramer. 181 pp.

Dawes, C. J. 1974. Marine Algae of the West Coast of Florida. University of Miami Press.

Dillard, G.E. 1989a. Freshwater algae of the Southeastern United States. Part 1. Chlorophyceae:Volvocales, Testrasporales, and Chlorococcales. Bibliotheca, 81.

Dillard, G.E. 1989b. Freshwater algae of the Southeastern United States. Part 2. Chlorophyceae:Ulotrichales, Microsporales, Cylindrocapsales, Sphaeropleales, Chaetophorales, Cladophorales,

Schizogoniales, Siphonales, and Oedogoniales. Bibliotheca Phycologica, 83.

Dillard, G.E. 1990. Freshwater algae of the Southeastern United States. Part 3. Chlorophyceae:Zygnematales: Zygenmataceae, Mesotaeniaceae, and Desmidaceae (Section 1). Bibliotheca

Phycologica, 85.

Dillard, G.E. 1991. Freshwater algae of the Southeastern United States. Part 4. Chlorophyceae:Zygnemateles: Desmidaceae (Section 2). Bibliotheca Phycologica, 89.


21/23




Drouet, F. 1968. Revision of the classification of the oscillatoriaceae. Monograph 15. Academy ofNatural Sciences, Philadelphia. Fulton Press, Lancaster, Pennsylvania.

Hohn, M.H. and J. Hellerman. 1963. The taxonomy and structure of diatom populations from threeNorth American rivers using three sampling methods. Transaction of the American MicroscopalSociety82:250-329.

Hustedt, F. 1927-1966. Die kieselalgen In Rabenhorsts Kryptogamen-flora von DeutschlandOsterreich und der Schweiz VII. Leipzig, West Germany.

Hustedt, F. 1930. Bacillariophyta (Diatomae). In Pascher, A. (ed). Die suswasser FloraMitteleuropas. (The freshwater flora of middle Europe). Gustav Fischer Verlag, Jena, Germany.

Jarrett, G.L. and J.M. King. 1989. The diatom flora (Bacillariphyceae) of Lake Barkley. U.S. ArmyCorps of Engineers, Nashville Dist. #DACW62-84-C-0085.

Krammer, K. and H. Lange-Bertalot. 1986-1991. Susswasserflora von Mitteleuropa. Band 2. Parts1-4. Bacillariophyceae. Gustav Fischer Verlag. Stuttgart. New York.

Lange-Bertalot, H. and R. Simonsen. 1978. A taxonomic revision of the Nitzschia lanceolataeGrunow: 2. European and related extra-European freshwater and brackish water taxa. Bacillaria1:11-111.

Lange-Bertalot, H. 1980. New species, combinations and synonyms in the genus Nitzschia.Bacillaria3:41-77.

Patrick, R. and C.W. Reimer. 1966. The diatoms of the United States, exclusive of Alaska andHawaii. Monograph No. 13. Academy of Natural Sciences, Philadelphia, Pennsylvania.

Patrick, R. and C.W. Reimer. 1975. The Diatoms of the United States. Vol. 2, Part 1. MonographNo. 13. Academy of Natural Sciences, Philadelphia, Pennsylvania.

Prescott, G.W. 1962. The algae of the Western Great Lakes area. Wm. C. Brown Co., Dubuque,Iowa.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1975. A Synopsis of North American desmids.Part II. Desmidaceae: Placodermae. Section 1. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1977. A synopsis of North American desmids.Part II. Desmidaceae: Placodermae. Section 2. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1981. A synopsis of North American desmids.Part II. Desmidaceae: Placodermae. Section 3. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W. 1978. How to know the freshwater algae. 3rd Edition. Wm. C. Brown Co.,Dubuque, Iowa.

Simonsen, R. 1987. Atlas and catalogue of the diatom types of Friedrich Hustedt. Vol. 1-3. J.Cramer. Berlin, Germany.


22/23



Smith, M. 1950. The Freshwater Algae of the United States. McGraw-Hill, New York, New York.

Taylor, W. R. 1960. Marine algae of the eastern tropical and subtropical coasts of the Americas.University of Michigan Press, Ann Arbor, Michigan.

VanLandingham, S. L. 1982. Guide to the identification, environmental requirements and pollution

tolerance of freshwater blue-green algae (Cyanophyta). EPA-600/3-82-073.

Whitford, L.A. and G.J. Schumacher. 1973. A manual of freshwater algae. Sparks Press, Raleigh,North Carolina.

Wujek, D.E. and R.F. Rupp. 1980. Diatoms of the Tittabawassee River, Michigan. BibliothecaPhycologia50:1-100.

6.4 AUTECOLOGICAL REFERENCES FOR PERIPHYTON

Beaver, J. 1981.Apparent ecological characteristics of some common freshwater diatoms. OntarioMinistry of the Environment. Rexdale, Ontario, Canada.

Cholnoky, B. J. 1968. kologie der Diatomeen in Binnegewssern. Cramer, Lehre.

Fabri, R. and L. Leclercq. 1984. Etude cologique des rivires du nord du massif Ardennais(Belgique): flore et vgtation de diatomees et physico-chimie des eaux. 1. Station scientifique desHautes Fagnes, Robertville. 379 pp.

Fjerdingstad, E. 1950. The microflora of the River Molleaa with special reference to the relation ofbenthic algae to pollution.Folia Limnologica Scandanavica5, 1-123.

Hustedt, F. 1938-39. Systamatische und kologische Untersuchungen ber die Diatomeen-Flora von

Java, Bali und Sumatra nach dem Material deter Deutschen Limnologischen Sunda-Expedition.Allgemeiner Teil. I. Ubersicht ber das Untersuchungsmaterial und Charakterisktik der Diatomeenflorader einzelnen Gebiete. II. Die Diatomeen flora der untersuchten Gesssertypen. III. Die kologischeFaktoren und ihr Einfluss auf die Diatomeenflora. Archiv fr Hydrobiologie, Supplement Band,15:638-790 (1938); 16:1-155 (1938); 16:274-394 (1939).

Hustedt, F. 1957. Die Diatomeenflora des Flusssystems der Weser im Gebiet der Hansestadt Bremen.Abhandlungen naturwissenschaftlichen. Verein zu Bremen, Bd. 34, Heft 3, S. 181-440, 1 Taf.

Lange-Bertalot, H. 1978. Diatomeen-Differentialarten anstelle von Leitformen: ein geeigneteresKriterium der Gewsserbelastung.Archiv fr Hydrobiologie Supplement 51, 393-427.

Lange-Bertalot, H. 1979. Pollution tolerance of diatoms as a criterion for water quality estimation.Nova Hedwigia64, 285-304.

LeCointe C., M. Coste, and J. Prygiel. 1993. "OMNIDIA" software for taxonomy, calculation ofdiatom indices and inventories management.Hydrobiologia269/270: 509-513.

Lowe, R. L. 1974.Environmental Requirements and Pollution Tolerance of Freshwater Diatoms. USEnvironmental Protection Agency, EPA-670/4-74-005. Cincinnati, Ohio, USA.


23/23


Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton Benthic

Palmer, C. M. 1969. A composite rating of algae tolerating organic pollution.Journal of Phycology5,78-82.

Rott, E., G. Hofmann, K. Pall, P. Pfister, and E. Pipp. 1997. Indikationslisten fr Aufwuchsalgen insterreichischen Fliessgewssern. Teil 1: Saprobielle Indikation. Wasserwirtschaftskataster.Bundesminsterium fr Land- und Forstwirtschaft. Stubenring 1, 1010 Wein, Austria.

Sldecek, V. 1973. System of water quality from the biological point of view. Archiv frHydrobiologie und Ergebnisse Limnologie7, 1-218.

Van Dam, H., Mertenes, A., and Sinkeldam, J. 1994. A coded checklist and ecological indicatorvalues of freshwater diatoms from the Netherlands. Netherlands Journal of Aquatic Ecology28,117-33.

Vanlandingham, S. L. 1982. Guide to the identification, environmental requirement and pollutiontolerance of freshwater blue-green algae (Cyanophyta). U. S. Environmental Protection Agency.EPA-600/3-82-073.

Watanabe, T., Asai, K., Houki, A. Tanaka, S., and Hizuka, T. 1986. Saprophilous and eurysaprobicdiatom taxa to organic water pollution and diatom assemblage index (DAIpo).Diatom 2:23-73.

USEPA 2006 Perifiton Protocolos

Documents

Transcript of USEPA 2006 Perifiton Protocolos